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Doctoral Thesis
DOI
10.11606/T.9.2015.tde-29042015-100916
Document
Author
Full name
Juliana Nunes da Cruz
E-mail
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2014
Supervisor
Committee
Nascimento, João Roberto Oliveira do (President)
Moreira, Vanessa
Hassimotto, Neuza Mariko Aymoto
Marquez, Ursula Maria Lanfer
Pimenta, Daniel Carvalho
Title in Portuguese
Hidrolisado proteico da semente de cupuaçu como fonte de peptídeos inibidores da enzima conversora da angiotensina I
Keywords in Portuguese
Atividade inibitória da ECA
Hidrolisado proteico
Peptídeos bioativos
Semente de cupuaçu
Abstract in Portuguese
Peptídeos com ação inibitória da enzima conversora da angiotensina I (ECA) podem ser obtidos a partir de diversos alimentos e exercer efeito anti-hipertensivo. O cupuaçu (Theobroma grandiflorum S.), fruto nativo da Amazônia, possui sementes comestíveis contendo proteína de reserva similar à do cacau (Theobroma cacao L.), as quais parecem ser fonte de peptídeos inibidores da ECA. Desse modo, o objetivo deste trabalho foi investigar in vitro a ocorrência de peptídeos inibidores da ECA no hidrolisado proteico da semente de cupuaçu obtido por ação da Alcalase. O hidrolisado revelou o desaparecimento de proteínas entre 27 a 180 kDa, incluindo as globulinas, e o surgimento daquelas abaixo de 15 kDa após 2 h de hidrólise, indicando a formação de peptídeos menores. O ensaio de atividade utilizando o substrato Abz-FRK(Dnp)-P-OH revelou que o hidrolisado total promoveu 65% de inibição da ECA e esse pool peptídico foi fracionado em cinco frações (F1-F5) por cromatografia em fase reversa (RP-HPLC). Após a etapa de purificação, o monitoramento da inibição apontou, ao final, duas frações (3.2.8 e 3.4.10) com maior atividade inibitória. Oito peptídeos foram identificados por LC-MS/MS, sendo três deles já conhecidos como inibidores da ECA. Outros cinco novos peptídeos identificados (FLEK, GSGKHVSP, LDNK, MVVDKLF e MEKHS) foram sintetizados e tiveram sua ação inibitória validada por ensaios in vitro. O peptídeo GSGKHVSP apresentou a menor IC50 (3,11 µM) e Ki (0,74 µM), sendo um inibidor tipo misto quanto ao seu mecanismo de inibição revelado pelo gráfico de Lineweaver-Burk. Os resultados permitem concluir que o isolado proteico da semente de cupuaçu pode ser uma fonte para obtenção de peptídeos anti-hipertensivos, a despeito de serem necessárias investigações sobre a resistência desses peptídeos à digestão gastrointestinal e a eficácia da inibição in vivo.
Title in English
Hydrolyzed cupuassu seed protein as a source of angiotensin I- converting enzyme inhibitory peptides
Keywords in English
ACE-inibitory activity
Bioactive peptides
Cupuassu seed
Protein hydrolysate
Abstract in English
Peptides with angiotensin I-converting enzyme (ACE) inhibitory activity may be obtained from several foods and cause antihypertensive effect. Cupuassu (Theobroma grandiflorum S.), a native fruit from Amazon, has edible seeds with a storage protein similar to that of cocoa (Theobroma cacao L.) which seems to have incrypted ACE inhibitor peptides. Thus, the aim of this project was to investigate the in vitro formation of ACE inhibitory peptides in protein hydrolysate from cupuassu seeds using Alcalase enzyme. The hydrolysate revealed the disappearance of proteins between 27 and 181 kDa after 2h hydrolysis, including the globulin, and the increase of those below 15 kDa, indicating the formation of peptides. The ACE inhibitory activity assays using the substrate Abz-FRK(Dnp)P-OH revealed the hydrolysate had 65% ACE inhibition and the pool of peptides was fractionated into five fractions (F1-F5) by reversed phase high-performance liquid chromatography (RP-HPLC). After the purification step, two fractions (3.2.8 e 3.4.10) exhibited the highest ACE-inhibitory activity. Eight peptides had been identified by LC-MS/MS and three of them were ACE inhibitors. The other newly identified peptides (FLEK, GSGKHVSP, LDNK, MVVDKLF and MEKHS) were synthesized and in vitro assayed for ACE inhibitory activity. The peptide GSGKHVSP had the lower IC50 (3.11 µM) and Ki (0.74 µM). Lineweaver-Burk plots suggest this peptide is a mixed-type inhibitor according to the inhibition mechanism. The results indicate that protein isolate from cupuassu seeds may be a good protein source of antihypertensive peptides and further investigation is needed in order to evaluate the resistance of these peptides to gastrointestinal digestion and the inhibitory activity in vivo.
 
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Publishing Date
2015-05-28
 
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